Method of screening antiplatelet agents

ABSTRACT

A screening tool for an antiplatelet agent wherein the tool is a human ADP receptor P2T AC  protein, a variation functionally equivalent thereto, or a homologous protein thereof, and a screening tool for an antiplatelet agent wherein the tool is a transformant which is transformed with an expression vector comprising a DNA encoding the protein and is expressing the polypeptide are disclosed. Further, a method for detecting an ADP receptor P2T AC  ligand, antagonist, or agonist by using the screening tool for an antiplatelet agent, and a method for screening an antiplatelet agent by the detecting method are disclosed.

TECHNICAL FIELD

The present invention relates to a method for screening antiplateletagents.

BACKGROUND ART

The platelet was discovered in 1842 by Donne [C. R. Acad. Sci.(paris),14, 336-368, 1842], and has long been regarded as a component of bloodnecessary for hemostasis. It is now known that the platelet plays notonly a main role in the hemostatic system, but also multifunctionalroles, for example, a clinically notable generation of arteriosclerosis,circulatory diseases including thrombotic diseases, metastasis ofcancer, inflammation, rejection after grafting, participation in immunereaction, or the like.

At present, revascularization by pharmacological or physical methods iscarried out to treat thrombotic diseases and ischemic diseases. However,it has been recently found that the activation, adhesion, and/oraggregation of the platelets is promoted by a collapse of blood vesseltissue including an endothelial cell after revascularization, or acollapse of a fibrinolysis-coagulation balance caused by a medicamentper se, which becomes a clinical problem. For example, it is known that,after revascularization by a thrombolytic therapy using t-PA or thelike, the fibrinolytic activity and/or coagulative activity areactivated, and then the systemic fibrinolysis-coagulation balancecollapses. Clinically, this causes re-occlusion, and becomes a criticalproblem therapeutically (J. Am. Coll. Cardiol. 12, 616-623, 1988).

In addition, the PTCA (Percutaneous transluminal coronary angioplasty)therapy has quickly become widely used, and has achieved good results inthe treatment of diseases based on aortostenosis or coronary stenosissuch as angina, myocardial infarction, or the like. However, the therapyinjures blood vessel tissue including an endothelial cell, and acutecoronary obstruction, and restenosis, which is observed in approximately30% of cases, become a problem.

The platelet plays an important role in these various thromboticdisorders (such as re-occlusion or the like) after the revascularizationtherapy. Therefore, an antiplatelet agent is desired as an agent fortreating or preventing these thrombotic disorders.

In this connection, adenosine 5′-diphosphate (ADP) is known as animportant factor which induces or promotes the activation, adhesion, andaggregation of the platelets. ADP is released from platelets activatedby collagen, thrombin, or the like, or from hemocytes, vascularendothelial cells, or organs injured by revascularization or the like.It is considered that ADP activates the platelets via a Gprotein-coupled ADP receptor P2T located in the platelet membrane(Biochem. J., 336, 513-523, 1998).

It has been suggested that a platelet ADP receptor P2T_(PLC) which iscoupled to Gq, one of the G proteins, and increases an intracellularCa²⁺ concentration via phospholipase C (PLC), and a platelet ADPreceptor P2T_(AC) which is coupled to Gi, one of the G proteins, andsuppresses an activity of adenylate cyclase (AC) are present as plateletADP receptors. At present, the platelet ADP receptor P2T_(PLC) has beenidentified as the receptor known as platelet ADP receptor P2Y1, but theentity of platelet ADP receptor P2T_(AC) is not identified (Kunapuli, S.P. et al., Trends Pharmacol. Sci., 19, 391-394, 1998).

It is considered that Ticlopidine or Clopidogrel used as an antiplateletagent functions by inhibiting the ADP receptor P2T_(AC) via itsmetabolite in a body (Savi, P. J., Pharmaclo. Exp. Ther., 269, 772-777,1994). Further, ARL67085, which is synthesized as a derivative ofadenosine triphosphate (ATP), which is an ADP receptor antagonist in abody, exhibits an activity of suppressing a platelet aggregation by theantagonist activity against the platelet ADP receptor P2T_(AC), and theeffectiveness thereof is proven by using a thrombosis model (Mills, D.C., Thromb. Hemost., 76, 835-856, 1996; and Humphries, R. G., TrendsPharmacol. Sci., 16, 179-181, 1995). Further, a derivative of Ap4A[P¹,P⁴-di(adenosine-5′)tetraphosphate] exhibits an activity suppressingthe platelet aggregation by ADP, by the antagonist activity against theplatelet ADP receptor P2T_(AC), and the effectiveness thereof is provenby using a thrombosis model (Kim, B. K., Proc. Natl. Acad. Sci. USA, 89,2370-2373, 1992).

From the above information, an antagonist against the platelet ADPreceptor P2T_(AC) is desired as a strong antiplatelet agent. However,Ticlopidine or Clopidogrel exhibits a weak antiplatelet activity, andhas problems such as a strong side effect or the like. Further, ARL67085or derivatives thereof (ATP analogues), derivatives of Ap4A, or thelike, which is studied as the ADP receptor antagonist, is a derivativeof nucleotide, and then an oral bioavailability is not sufficient, andfurther problems arise such as a weak activity of suppressing theplatelet aggregation. Therefore, an ADP receptor antagonist having astrong oral bioavailability is intensely desired (CAPRIE STEERINGCOMMITTEE, Lancet, 348, 1329-1339, 1996).

However, the ADP receptor P2T_(AC) protein has not been identified asyet. Therefore, it is difficult to construct a convenient system forscreening such a compound, and farther, the development of the ADPreceptor P2T_(AC) antagonist has not progressed.

In this connection, with regard to a DNA encoding a polypeptideconsisting of the same amino acid sequence as that of a human ADPreceptor P2T_(AC) protein, which may be used in the present invention,and an amino acid sequence deduced from the DNA, there are severalreports (WO00/22131, WO00/31258, WO00/28028, and WO98/50549 pamphlets).However, ligands are not elucidated in the reports, and no reportsdisclose that the protein is an ADP receptor located in the platelet.

DISCLOSURE OF INVENTION

Therefore, the object of the present invention is to provide aconvenient screening system to obtain an adenosine diphosphate (ADP)receptor P2T_(AC) antagonist which is useful as an antiplatelet agent,and a novel antiplatelet agent.

With the aim of solving the aforementioned problems, the presentinventors have conducted intensive studies and, as a result,successfully isolated a nucleic acid (more particularly, an HORK3 gene)encoding the P2T_(AC) receptor, and have determined the nucleotidesequence thereof and the deduced amino acid sequence. Further, theinventors prepared a vector comprising the nucleic acid encoding thereceptor, and a host cell comprising the vector, and made it possible toproduce a novel recombinant P2T_(AC) receptor by expressing the P2T_(AC)receptor by the use of the host cell. The inventors confirmed that thereceptor exhibited an ADP receptor P2T_(AC) activity, and thus thereceptor and the cell expressing the receptor could be used as ascreening tool for an antiplatelet agent. The inventors established amethod for detecting whether or not a test compound is an ADP receptorP2T_(AC) ligand, antagonist, or agonist by using the receptor or thecell expressing the receptor, and a method for screening an antiplateletagent by using the detecting method. The inventors confirmed thatcompounds known to exhibit an antiplatelet activity (more particularly2MeSAMP or AR-C69931MX) exhibited an antagonist activity of thereceptor, by using the detecting method, and showed that an antagonistof the receptor was certainly useful as an antiplatelet agent. Further,the inventors established a process for manufacturing a pharmaceuticalcomposition for antiplatelet, comprising the detecting step, andcompleted the present invention.

Namely, the present invention relates to:

-   [1] a screening tool for an antiplatelet agent, wherein the tool is-   (1) a polypeptide (hereinafter sometimes referred to as “human ADP    receptor P2T_(AC) protein”) having an amino acid sequence of SEQ ID    NO: 2, or-   (2) a polypeptide (hereinafter referred to as “variation    functionally equivalent”) having an amino acid sequence in which one    or plural amino acids are deleted, substituted, and/or added at one    or plural positions in an amino acid sequence of SEQ ID NO: 2, and    exhibiting an activity (hereinafter referred to as “ADP receptor    P2T_(AC) activity”) of suppressing an adenylate cyclase activity by    binding to ADP and coupling with Gi:-   [2] a screening tool for an antiplatelet agent, wherein the tool is    a polypeptide (hereinafter referred to as “homologous protein”)    having an amino acid sequence having a 90% or more homology with an    amino acid sequence of SEQ ID NO: 2, and exhibiting an activity of    suppressing an adenylate cyclase activity by binding to ADP and    coupling with Gi (hereinafter the screening tools of the items [1]    and [2] for an antiplatelet agent are collectively referred to as    “polypeptide-type screening tool for an antiplatelet agent”);-   [3] a screening tool for an antiplatelet agent, wherein the tool is    a transformant which is transformed with an expression vector    comprising a DNA encoding (1) a polypeptide having an amino acid    sequence of SEQ ID NO: 2 (i.e., human ADP receptor P2T_(AC) protein)    or (2) a polypeptide having an amino acid sequence in which one or    plural amino acids are deleted, substituted, and/or added at one or    plural positions in an amino acid sequence of SEQ ID NO: 2, and    exhibiting an activity of suppressing an adenylate cyclase activity    by binding to ADP and coupling with Gi (i.e., variation functionally    equivalent), and is expressing the polypeptide;-   [4] a screening tool for an antiplatelet agent, wherein the tool is    a transformant which is transformed with an expression vector    comprising a DNA encoding a polypeptide having an amino acid    sequence having a 90% or more homology with an amino acid sequence    of SEQ ID NO: 2, and exhibiting an activity of suppressing an    adenylate cyclase activity by binding to ADP and coupling with Gi    (i.e., homologous protein), and is expressing the polypeptide    (hereinafter the screening tools of the items [3] and [4] for an    antiplatelet agent are collectively referred to as    “transformant-type screening tool for an antiplatelet agent”);-   [5] a method for detecting whether or not a compound to be tested is    an ADP receptor P2T_(AC) ligand, comprising the steps of:-   bringing a polypeptide of the item [1] or [2] (i.e., the human ADP    receptor P2T_(AC) protein, variation functionally equivalent, or    homologous protein), a cell membrane fraction comprising the    polypeptide, or a transformant of the item [3] or [4] into contact    with the compound to be tested, in the presence of a labeled ligand    of an ADP receptor P2T_(AC), and-   analyzing a change of an amount of the labeled ligand which binds to    the polypeptide, the cell membrane fraction, or the transformant    (hereinafter referred to as “ligand detecting method”);-   [6] a method for detecting whether or not a compound to be tested is    an ADP receptor P2T_(AC) antagonist or agonist, comprising the steps    of:-   bringing the compound to be tested into contact with a transformant    of the item [3] or [4] which is co-expressing a G protein chimera    comprising a polypeptide fragment having an activity promoting a    phospholipase C activity of a G protein promoting the phospholipase    C activity and a polypeptide fragment having a receptor-coupled    activity of Gi, said G protein chimera having an amino acid sequence    of SEQ ID NO: 11 at the C-terminus; and-   analyzing a change of an intracellular Ca²⁺ concentration in the    transformant (hereinafter referred to as “Ca²⁺-type detecting    method”);-   [7] a method for detecting whether or not a compound to be tested is    an ADP receptor P2T_(AC) antagonist or agonist, comprising the steps    of:-   bringing a transformant of the item [3] or [4] into contact with the    compound to be tested, in the presence of a platelet ADP receptor    P2T_(AC) agonist, and-   analyzing a change of an intracellular cAMP concentration in the    transformant (hereinafter referred to as “cAMP-type detecting    method”);-   [8] a method for screening an antiplatelet agent, comprising the    steps of:-   detecting whether or not a compound to be tested is an ADP receptor    P2T_(AC) ligand, antagonist, or agonist by the ligand detecting    method, the Ca²⁺-type detecting method, or the cAMP-type detecting    method, or a combination thereof, and selecting the ADP receptor    P2T_(AC) antagonist; and-   [9] a process for manufacturing a pharmaceutical composition for    antiplatelet, comprising the steps of:-   detecting whether or not a compound to be tested is an ADP receptor    P2T_(AC) ligand, antagonist, or agonist by the ligand detecting    method, the Ca²⁺-type detecting method, or the cAMP-type detecting    method, or a combination thereof, and preparing a medicament.

The “Gi” is a subfamily of the G proteins which are coupled to areceptor and function as a signal transduction and amplification factorinto a cell, and a G protein which suppresses an adenylate cyclaseactivity. When the adenylate cyclase activity is suppressed, forexample, an intracellular cAMP concentration decreases.

The “G protein promoting the phospholipase C activity” is a subfamily ofG proteins which are coupled to a receptor and function as a signaltransduction and amplification factor into a cell, and a G protein whichpromotes a phospholipase C activity. When the phospholipase C activityis induced, for example, an intracellular Ca²⁺ concentration increases.As the G protein promoting the phospholipase C activity, there may bementioned, for example, Gq.

The “ADP receptor P2T_(AC)” means a polypeptide having the ADP receptorP2T_(AC).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the effect of 2MeSAMP in an ADP-inducedplatelet aggregation of platelet-rich plasma (PRP) derived from humanblood-treated with sodium citrate.

FIG. 2 is a graph showing the effect of AR-C69931MX in an ADP-inducedplatelet aggregation of PRP derived from human blood-treated with sodiumcitrate.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail hereinafter.

(1) The Screening Tool for an Antiplatelet Agent

The screening tool of the present invention for an antiplatelet agentincludes a polypeptide-type screening tool for an antiplatelet agent anda transformant-type screening tool for an antiplatelet agent.

1) The Polypeptide-type Screening Tool for an Antiplatelet Agent

The polypeptide-type screening tool of the present invention for anantiplatelet agent includes

-   (i) a screening tool for an antiplatelet agent, wherein the tool is    the human ADP receptor P2T_(AC) protein, i.e., the polypeptide    having the amino acid sequence of SEQ ID NO: 2;-   (ii) a screening tool for an antiplatelet agent, wherein the tool is    a variation functionally equivalent, i.e., a polypeptide having an    amino acid sequence in which one or plural amino acids are deleted,    substituted, and/or added at one or plural positions in the amino    acid sequence of SEQ ID NO: 2, and exhibiting the ADP receptor    P2T_(AC) activity; and-   (iii) a screening tool for an antiplatelet agent, wherein the tool    is a homologous protein, i.e., a polypeptide having an amino acid    sequence having a 90% or more homology with the amino acid sequence    of SEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC) activity.

The polypeptide having the amino acid sequence of SEQ ID NO: 2, whichmay be used as the polypeptide-type screening tool of the presentinvention for an antiplatelet agent, is a human ADP receptor P2T_(AC)protein consisting of 342 amino acid residues. Although it has beensuggested that the ADP receptor P2T_(AC) protein exists in platelets,the entity thereof had not been identified until the present inventorsfirst elucidated its function.

Then, it was first published in a reference after the priority date ofthe present application that the polypeptide having the amino acidsequence of SEQ ID NO: 2 is a platelet ADP receptor [Nature, 409, Jan.11, 2001, 202-207; J. B. C., 276, (11), March 16, 8608-8615, 2001;WO01/46454 pamphlet]. In the basic patent application of the priority ofthe WO01/46454 pamphlet, although a rat ADP receptor gene was cloned andthe sequence thereof was determined, as to a human ADP receptor gene,only a clone containing a partial sequence was obtained and the sequencethereof was determined. Therefore, it was first accomplished by thepresent applicant that the human ADP receptor P2T_(AC) protein and thepolynucleotide encoding the protein was obtained, the function thereofwas elucidated, and a patent application relating to the invention ofthe method for screening an antiplatelet agent using them was filed.

The variation functionally equivalent which may be used as thepolypeptide-type screening tool of the present invention for anantiplatelet agent is not particularly limited, so long as it is apolypeptide having an amino acid sequence in which one or plural(preferably 1 to 10, more preferably 1 to 5) amino acids are deleted,substituted, and/or added at one or plural positions in the amino acidsequence of SEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC)activity. Further, an origin of the variation functionally equivalent isnot limited to a human.

The variation functionally equivalent includes, for example, humanvariations of the human ADP receptor P2T_(AC), and ADP receptorsP2T_(AC) derived from organisms other than a human (such as a mouse, arat, a hamster, or a dog) or variations thereof, and further proteinsobtained by artificially modifying these native proteins (i.e., humanvariations, or ADP receptors P2T_(AC) derived from organisms other thana human or variations thereof) or the human ADP receptor P2T_(AC) bygenetic engineering techniques. The term “variation” as used hereinmeans an individual difference between the same proteins in the samespecies or a difference between homologous proteins in several species.

Human variations of the human ADP receptor P2T_(AC), or ADP receptorsP2T_(AC) derived from organisms other than a human or variations thereofmay be obtained by those skilled in the art based on the information ofa nucleotide sequence (for example, the nucleotide sequence of SEQ IDNO: 1) of the human ADP receptor P2T_(AC) gene. In this connection,genetic engineering techniques may be performed in accordance with knownmethods (for example, Maniatis, T. et al., “Molecular Cloning—ALaboratory Manual”, Cold Spring Harbor Laboratory, NY, 1982, or thelike), unless otherwise specified.

For example, an appropriate probe or appropriate primers are designed inaccordance with the information of a base sequence of the human ADPreceptor P2T_(AC) gene. A PCR method or a hybridization method iscarried out using a sample (for example, total RNA or an mRNA fraction,a cDNA library, or a phage library) derived from an organism (forexample, a mammal such as a human, a mouse, a rat, a hamster, or a dog)of interest and the primers or the probe to obtain a gene encoding theprotein. A desired protein can be obtained by expressing the resultinggene in an appropriate expression system and confirming that theexpressed protein suppresses the adenylate cyclase activity by bindingto ADP and coupling with Gi, for example, by the method described inExample 3 or 4.

Further, the protein artificially modified by genetic engineeringtechniques can be obtained by, for example, the following procedure. Agene encoding the protein is obtained by a conventional method such assite-specific mutagenesis (Mark, D. F. et al., Proc. Natl. Acad. Sci.USA, 81, 5662-5666, 1984). A desired protein can be obtained byexpressing the resulting gene in an appropriate expression system andconfirming that the expressed protein suppresses the adenylate cyclaseactivity by binding to ADP and coupling with Gi, for example, by themethod described in Example 3 or 4.

The homologous protein which may be used as the polypeptide-typescreening tool of the present invention for an antiplatelet agent is notparticularly limited, so long as it is a polypeptide having an aminoacid sequence having a 90% or more homology with the amino acid sequenceof SEQ ID NO: 2, and exhibiting the ADP receptor P2T_(AC) activity. Thehomologous protein may have an amino acid sequence having preferably a95% or more homology, more preferably a 98% or more homology, mostpreferably a 99% or more homology, with respect to the amino acidsequence of SEQ ID NO: 2.

The term “homology” as used herein means a value which can be obtainedby a BLAST package [Basic local alignment search tool; Altschul, S. F.et al., J. Mol. Biol., 215, 403-410, (1990)]. The homology in the aminoacid sequence can be calculated by a BLAST search algorithm. Moreparticularly, this value can be obtained by using a bl2seq program(Tatiana A. Tatusova and Thomas L. Madden, FEMS Microbiol. Lett., 174,247-250, 1999) with a default parameter in a BLAST package (sgi32bitedition, version 2.0.12; obtained from NCBI). Default parameters of thebl2seq program include “blastp” as a search program, “0” as a cost toopen a gap, “0” as a cost to extend a gap, “SEG” as a filter of thequery sequence, and “BLOSUM62” as a matrix.

The polypeptides which may be used as the polypeptide-type screeningtool of the present invention for an antiplatelet agent (i.e., the humanADP receptor P2T_(AC) protein, the variations functionally equivalent,and the homologous proteins; hereinafter referred to as “polypeptide fora screening tool”) may be obtained by various known methods, such asknown genetic engineering techniques using a gene encoding a protein ofinterest. More particularly, the polypeptide for a screening tool may beprepared by culturing a transformant described below (i.e., atransformant which is transformed with an expression vector comprising aDNA encoding the polypeptide for a screening tool and expressing thepolypeptide) under a condition in which an expression of the polypeptidefor a screening tool may be performed, and separating and purifying theprotein of interest from the resulting culture by commonly used methodsfor a separation and a purification of receptor proteins.

When the polypeptide for a screening tool is prepared, the method forobtaining the gene encoding the polypeptide is not particularly limited.For example, when the human ADP receptor P2T_(AC) protein is prepared,for example, the DNA consisting of the nucleotide sequence of SEQ ID NO:1 may be used as the gene encoding the protein. In this connection,codons for each amino acid are known and can be optionally selected anddetermined by the conventional method, for example, by taking a codonusage of each host to be used into consideration (Crantham, R. et al.,Nucleic Acids Res., 9, r43-r74, 1981).

The DNA consisting of the nucleotide sequence of SEQ ID NO: 1 may beobtained by, for example, ligating DNA fragments prepared by a chemicalsynthesis method, or a polymerase chain reaction (PCR) method (Saiki, R.K. et al., Science, 239, 487-491, 1988) using a cDNA library derivedfrom a cell or tissue capable of producing the human ADP receptorP2T_(AC) protein as a template and an appropriate primer set designed inaccordance with the nucleotide sequence of SEQ ID NO: 1. As the cell ortissue capable of producing the human ADP receptor P2T_(AC) protein,there may be mentioned, for example, human platelets, the human brain,or the like. As the primer set, there may be mentioned, for example, acombination of an oligonucleotide consisting of the nucleotide sequenceof SEQ ID NO: 3 and an oligonucleotide consisting of the nucleotidesequence of SEQ ID NO: 4.

A separation and purification method which may be used for preparing thepolypeptide for a screening tool is not particularly limited, but can beperformed, for example, in accordance with the following procedure. Forexample, a cell membrane fraction containing the polypeptide for ascreening tool can be obtained by culturing cells expressing thepolypeptide for a screening tool on the surface thereof, suspending thecultured cells in a buffer, homogenizing the suspension, andcentrifuging the homogenate. After the resulting cell membrane fractionis solubilized, the polypeptide for a screening tool can be purified bytreating the mixture with a commonly used treatment with a proteinprecipitant, ultrafiltration, various liquid chromatography techniquessuch as molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, affinity chromatography, orhigh performance liquid chromatography (HPLC), or dialysis, or acombination thereof. In this connection, when the cell membrane fractionis solubilized, characteristics of the receptor can be maintained afterthe solubilization, by using as mild as possible a solubilizing agent(such as CHAPS, Triton X-100, digitonin or the like).

When the polypeptide for a screening tool is prepared, a confirmation ofthe expression of the polypeptide, a confirmation of intracellularlocalization thereof, a purification thereof, or the like may be easilycarried out by expressing the polypeptide for a screening tool as afusion protein with an appropriate marker sequence in frame, ifnecessary. As the marker sequence, there may be mentioned, for example,a FLAG epitope, a hexa-histidine tag, a hemagglutinin tag, a mycepitope, or the like. Further, by inserting a specific sequencerecognized by a protease such as enterokinase, factor Xa, thrombin, orthe like between the marker sequence and the polypeptide for a screeningtool, the marker sequence may be removed by the protease. For example,there is a report in which a muscarinic acetylcholine receptor and ahexa-histidine tag were connected by a thrombin recognition sequence(Hayashi, M. K. and Haga, T., J. Biochem., 120, 1232-1238, 1996).

2) The Transformant-type Screening Tool for an Antiplatelet Agent

The transformant-type screening tool of the present invention for anantiplatelet agent includes

-   (i) a screening tool for an antiplatelet agent, wherein the tool is    a transformant which is transformed with an expression vector    comprising a DNA encoding the human ADP receptor P2T_(AC) protein    and is expressing the polypeptide;-   (ii) a screening tool for an antiplatelet agent, wherein the tool is    a transformant which is transformed with an expression vector    comprising a DNA encoding the variation functionally equivalent and    is expressing the polypeptide; and-   (iii) a screening tool for an antiplatelet agent, wherein the tool    is a transformant which is transformed with an expression vector    comprising a DNA encoding the homologous protein and is expressing    the polypeptide.

A host cell which may be used for preparing the transformants(hereinafter referred to as “transformant for a screening tool”) whichmay be used as the transformant-type screening tool of the presentinvention for an antiplatelet agent is not particularly limited, so longas the polypeptide for a screening tool can be expressed. As the hostcell, there may be mentioned, for example, commonly used knownmicroorganisms, such as Escherichia coli or Saccharomyces cerevisiae, orknown cultured cells, such as vertebral cells (for example, a CHO cell,HEK293 cell, or COS cell) or insect cells (for example, an Sf9 cell). Asthe vertebral cell, there may be mentioned, for example, a COS cell as asimian cell (Gluzman, Y., Cell, 23, 175-182, 1981), a dihydrofolatereductase defective strain of a Chinese hamster ovary cell (CHO)(Urlaub, G. and Chasin, L. A., Proc. Natl. Acad. Sci. USA, 77,4216-4220, 1980), a human embryonic kidney derived HEK293 cell, or a293-EBNA cell (Invitrogen) obtained by introducing an EBNA-1 gene ofEpstein Barr Virus into the HEK293 cell.

An expression vector which may be used for preparing the transformantfor a screening tool is not particularly limited, so long as thepolypeptide for a screening tool can be expressed. An appropriateexpression vector can be selected in accordance with a host cell to beused.

As an expression vector for a vertebral cell, a vector containing apromoter positioned upstream of a gene to be expressed, an RNA splicingsite, a polyadenylation site, a transcription termination sequence, andthe like may be generally used. The vector may further contain areplication origin, if necessary. As the expression vector, there may bementioned, for example, pSV2dhfr containing an SV40 early promoter(Subramani, S. et al., Mol. Cell. Biol., 1, 854-864, 1981), pEF-BOScontaining a human elongation factor promoter (Mizushima, S. and Nagata,S., Nucleic Acids Res., 18,5322, 1990), pCEP4 containing acytomegalovirus promoter (Invitrogen), or the like.

More particularly, when the COS cell is used as the host cell, a vectorhaving an SV40 replication origin, capable of performing an autonomousreplication in the COS cell, and having a transcription promoter, atranscription termination signal, and an RNA splicing site, may be usedas the expression vector. As the vector, there may be mentioned, forexample, pME18S (Maruyama, K. and Takebe, Y., Med. Immunol., 20, 27-32,1990), pEF-BOS (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18,5322, 1990), pCDM8 (Seed, B., Nature, 329, 840-842, 1987), or the like.

The expression vector may be incorporated into COS cells by, forexample, a DEAE-dextran method (Luthman, H. and Magnusson, G., NucleicAcids Res., 11, 1295-1308, 1983), a calcium phosphate-DNAco-precipitation method (Graham, F. L. and van der Ed, A. J., Virology,52, 456-457, 1973), a method using a cationic liposome reagent(Lipofectamine; Gibco BRL), an electroporation method (Neumann, E. etal., EMBO J., 1, 841-845, 1982), or the like.

When the CHO cell is used as the host cell, a transformant capable ofstably producing the polypeptide for a screening tool can be obtained bycarrying out a co-transfection of an expression vector comprising theDNA encoding the polypeptide for a screening tool, together with avector capable of expressing a neo gene which functions as a G418resistance marker, such as pRSVneo (Sambrook, J. et al., “MolecularCloning—A Laboratory Manual”, Cold Spring Harbor Laboratory, NY, 1989),pSV2-neo (Southern, P. J. and Berg, P., J. Mol. Appl. Genet., 1,327-341,1982), or the like, and selecting a G418 resistant colony.

When the 293-EBNA cell is used as the host cell, for example, pCEP4(Invitrogen) containing a replication origin of Epstein Barr Virus andcapable of performing an autonomous replication in the 293-EBNA cell, orthe like may be used as the expression vector.

The transformant for a screening tool may be cultured in accordance witha conventional method, and the polypeptide for a screening tool isproduced in the cell or on the cell surface. As a medium to be used inthe culturing, a medium commonly used in a selected host cell may beappropriately selected. For example, in the case of the COS cell, forexample, a medium such as an RPMI-1640 medium, a Dulbecco's modifiedEagle's minimum essential medium (DMEM), or the like may be used, bysupplementing it with a serum component such as fetal bovine serum (FBS)or the like if necessary. In the case of the 293-EBNA cell, a mediumsuch as a Dulbecco's modified Eagle's minimum essential medium (DMEM) orthe like with a serum component such as fetal bovine serum (FBS) or thelike and G418 may be used.

The transformant for a screening tool is not particularly limited, solong as the polypeptide for a screening tool is expressed. As thetransformant for a screening tool, it is preferable to express a Gprotein in which the amino acid sequence at the C-terminus is that ofSEQ ID NO: 11 (Asp-Cys-Gly-Leu-Phe), in addition to the polypeptide fora screening tool. The amino acid sequence of SEQ ID NO: 11 is thatconsisting of five amino acid residues at the C-terminus of Gi.Hereinafter the “G protein in which the amino acid sequence at theC-terminus is that of SEQ ID NO: 11” will be referred to as “C-terminusGi-type G protein”.

As the C-terminus Gi-type G protein, there may be mentioned, forexample,

-   (1) Gi, or-   (2) a G protein chimera which comprises a polypeptide fragment    having the activity promoting a phospholipase C activity of a G    protein (such as Gq) promoting the phospholipase C activity and a    polypeptide fragment having a receptor-coupled activity of Gi, and    which further has an amino acid sequence of SEQ ID NO: 11 at the    C-terminus. Hereinafter, the G protein chimera comprising a    polypeptide fragment having the activity of promoting a    phospholipase C activity and a polypeptide fragment having a    receptor-coupled activity of Gi will be referred to as Gqi.

The polypeptide for a screening tool recognizes the amino acid sequenceconsisting of five amino acid residues at the C-terminus of Gi (i.e.,the amino acid sequence of SEQ ID NO: 11), and binds to Gi. Therefore,the polypeptide for a screening tool may be bound to not only Gi, butalso Gqi. When the polypeptide for a screening tool and the C-terminusGi-type G protein are expressed in the transformant for a screeningtool, these polypeptides can bind to each other in the cell.

The “polypeptide fragment having the activity of promoting aphospholipase C activity of a Gq” is not particularly limited, so longas it does not comprise a C-terminal amino acid sequence which isnecessary to bind to a Gq-coupled platelet ADP receptor P2T_(PLC), andexhibits the activity of promoting the phospholipase C activity. Theremay be mentioned, for example, a polypeptide fragment at the N-terminalside of Gq in which the amino acid sequence consisting of five aminoacid residues at the C-terminus is deleted.

The “polypeptide fragment having a receptor-coupled activity of Gi” isnot particularly limited, so long as it comprises the amino acidsequence consisting of five amino acid residues at the C-terminus of Gi,and does not exhibit an activity suppressing the adenylate cyclaseactivity. There may be mentioned, for example, a polypeptide fragment atthe C-terminal side of Gi, consisting of the amino acid sequence of SEQID NO: 11.

(2) The Detecting Method for an ADP Receptor P2T_(AC) Ligand,Antagonist, or Agonist

It can be detected whether or not a test compound is an ADP receptorP2T_(AC) ligand, antagonist, or agonist, by using the polypeptide for ascreening tool or the transformant for a screening tool as a detectingtool.

The method of the present invention for detecting whether or not a testcompound is an ADP receptor P2T_(AC) ligand, antagonist, or agonistincludes

-   1) a method for detecting whether or not a test compound is a ligand    against the platelet ADP receptor P2T_(AC) (i.e., ligand detecting    method);-   2) a method for detecting whether or not a test compound is an    antagonist or agonist against the platelet ADP receptor P2T_(AC), by    the use of changes of an intracellular Ca²⁺ concentration in the    transformant as an indicator (i.e., Ca²⁺-type detecting method);-   3) a method for detecting whether or not a test compound is an    antagonist or agonist against the platelet ADP receptor P2T_(AC), by    the use of changes of an intracellular cAMP content in the    transformant as an indicator (i.e., cAMP-type detecting method); and-   4) a method for detecting whether or not a test compound is an    antagonist or agonist against the platelet ADP receptor P2T_(AC), by    the use of a GTPγS binding method (i.e., GTPγS binding-type    detecting method).

These detecting methods will be explained in this order hereinafter.

1) The Ligand Detecting Method

The ligand detecting method of the present invention is not particularlylimited, so long as

-   (i) the human ADP receptor P2T_(AC) protein, variation functionally    equivalent, or homologous protein (hereinafter referred to as    “polypeptide for a detecting tool”), a cell membrane fraction    comprising the polypeptide for a detecting tool, or a transformant    expressing the polypeptide for a detecting tool (hereinafter the    polypeptide for a detecting tool, the cell membrane fraction, and    the transformant are collectively referred to as “ligand-detecting    tool”) and-   (ii) a labeled ligand are used. It may be carried out, for example,    in accordance with the following procedure.

The ligand-detecting tool is prepared. Assay conditions (for example, abuffer to be used and the concentration thereof, ions to be added to thebuffer and the concentration thereof, and the pH in the assay system)are optimized. The ligand-detecting tool and a labeled ligand areincubated in the optimized buffer, together with a test compound, for apredetermined time. As the labeled ligand, for example, [³H]2-methylthio-ADP (2MeSADP) may be used. After the reaction, the whole isfiltered with a glass filter or the like, and the filter is washed withan appropriate volume of the buffer. The remaining radioactivity on thefilter is measured by a liquid scintillation counter or the like. It maybe detected whether or not the test compound is a ligand against the ADPreceptor P2T_(AC), by the obtained binding inhibition of the radioactiveligand as an indicator. More particularly, when the remainingradioactivity on the filter in the presence of the test compound islower than that in the absence of the test compound, it may be decidedthat the test compound is a ligand against the ADP receptor P2T_(AC). Itmay be carried out, for example, under the conditions described inExample 6.

2) The Ca²⁺-type Detecting Method

In the Ca²⁺-type detecting method of the present invention, atransformant (hereinafter referred to as transformant for Ca²⁺-typedetection) co-expressing

-   (i) the polypeptide for a detecting tool and-   (ii) a G protein chimera (such as Gqi) which comprises a polypeptide    fragment having the activity promoting a phospholipase C activity of    a G protein promoting the phospholipase C activity and a polypeptide    fragment having a receptor-coupled activity of Gi, and which further    has an amino acid sequence of SEQ ID NO: 11 at the C-terminus is    used as the transformant. As to the transformant, it is preferable    to use a cell in which an intracellular Ca²⁺ concentration is not    increased by ADP, as a host cell before the transformation. As the    host cell, there may be mentioned, for example, C6-15, one of rat    glioma cell lines (Change, K. et al., J. Biol. Chem., 270,    26152-26158, 1995).

In the case of detecting whether or not a test compound is an agonist inthe Ca²⁺-type detecting method of the present invention, thetransformant for Ca²⁺-type detection is brought into contact with a testcompound, and then changes of the intracellular Ca²⁺ concentration inthe transformant for Ca²⁺-type detection are analyzed (i.e., measured ordetected) directly or indirectly. Changes of the Ca²⁺ concentration maybe, for example, directly analyzed by the use of a calcium-bindingfluorescence reagent (such as fura2, fluo3, or the like), or indirectlyanalyzed by analyzing a transcriptional activity of a gene [such as agene obtained by introducing an activator protein 1 (AP1) responsivesequence upstream of a luciferase gene] in which a regulation of thetranscription is dependent on the Ca²⁺ concentration.

When the transformant for Ca²⁺-type detection is brought into contactwith a test compound, and then the intracellular Ca²⁺ concentrationtherein increases, it may be decided that the test compound is anagonist against the ADP receptor P2T_(AC). In this connection, as acontrol, a similar procedure is carried out using a control transformantnot expressing the polypeptide for a detecting tool but expressing Gqi,or a host cell before the transformation, instead of the transformantfor Ca²⁺-type detection co-expressing the polypeptide for a detectingtool and Gqi, and then it is preferable to confirm that theintracellular Ca²⁺ concentration in the control transformant or hostcell is not increased by the test compound.

In the case of detecting whether or not a test compound is an antagonistin the Ca²⁺-type detecting method of the present invention, thetransformant for Ca²⁺-type detection is brought into contact with a testcompound in the presence of a platelet ADP receptor P2T_(AC) agonist(such as 2MeSADP or ADP), and then changes of the intracellular Ca²⁺concentration therein are analyzed (i.e., measured or detected) directlyor indirectly. When the transformant for Ca²⁺-type detection is broughtinto contact with a test compound in the presence of a platelet ADPreceptor P2T_(AC) agonist, and then the increase in the intracellularCa²⁺ concentration therein by the agonist is inhibited or suppressed bythe test compound, it may be decided that the test compound is anantagonist against the ADP receptor P2T_(AC). This may be carried out,for example, under the conditions described in Example 3.

In this connection, as a control, the transformant for Ca²⁺ detection isbrought into contact with the platelet ADP receptor P2T_(AC) agonist inthe absence of a test compound, and then it is necessary to confirm adegree of the increase in the intracellular Ca²⁺ concentration thereinby the agonist.

As described above, in the Ca²⁺-type detecting method of the presentinvention, Gi per se is not used as the coupled protein, but Gqi isused. Therefore, it is possible to detect whether or not a test compoundis an antagonist or agonist by analyzing the Ca²⁺ concentration, not thecAMP concentration. In general, measurement can be carried out moreeasily and rapidly by using the Ca²⁺ concentration, in comparison withthe cAMP concentration.

3) The cAMP-type Detecting Method

In the cAMP-type detecting method of the present invention, atransformant expressing the polypeptide for a detecting tool(hereinafter referred to as “transformant for cAMP-type detection”) isused as the transformant. Gi is constitutively expressed in a commonlyused host cell, and thus the transformant for cAMP-type detection may beobtained by transforming a host cell with an expression vectorcomprising a DNA encoding the polypeptide for a detecting tool. As tothe transformant, it is preferable to use a cell in which anintracellular cAMP concentration is not decreased by ADP, as a host cellbefore the transformation. As the host cell, there may be mentioned, forexample, a CHO cell.

In the case of detecting whether or not a test compound is an agonist inthe cAMP-type detecting method of the present invention, thetransformant for cAMP-type detection is brought into contact with a testcompound, and then changes of the intracellular cAMP concentration inthe transformant for cAMP-type detection are analyzed (i.e., measured ordetected) directly or indirectly. It is preferable that the transformantfor cAMP-type detection is brought into contact with a test compound inthe presence of a compound (such as forskolin) capable of increasing thecAMP concentration.

Changes of the cAMP concentration may be, for example, directly analyzedby the use of a commercially available cAMP measuring kit (Amersham orthe like), or indirectly analyzed by analyzing a transcriptionalactivity of a gene [such as a gene obtained by introducing a cAMPresponsive element (CRE) upstream of a luciferase gene] in which aregulation of the transcription is dependent on the cAMP concentration.

When the transformant for cAMP-type detection is brought into contactwith a test compound, and then the intracellular cAMP concentrationtherein decreases, it may be decided that the test compound is anagonist against the ADP receptor P2T_(AC). In this case, the decrease inthe cAMP concentration by a test compound may be easily decided, when acompound (such as forskolin) capable of increasing the cAMPconcentration coexists. In this connection, as a control, a similarprocedure is carried out using a host cell not expressing thepolypeptide for a detecting tool, instead of the transformant forcAMP-type detection expressing the polypeptide for a detecting tool andGi, and then it is preferable to confirm that the intracellular cAMPconcentration in the host cell is not decreased by the test compound.

In the case of detecting whether or not a test compound is an antagonistin the cAMP-type detecting method of the present invention, thetransformant for cAMP-type detection is brought into contact with a testcompound in the presence of a platelet ADP receptor P2T_(AC) agonist(such as 2MeSADP or ADP), and then changes of the intracellular cAMPconcentration therein are analyzed (i.e., measured or detected) directlyor indirectly. When the transformant for cAMP-type detection is broughtinto contact with a test compound in the presence of a platelet ADPreceptor P2T_(AC) agonist (such as 2MeSADP or ADP), and then thedecrease in the intracellular cAMP concentration therein by the agonistis inhibited or suppressed by the test compound, it may be decided thatthe test compound is an antagonist against the ADP receptor P2T_(AC). Inthis case, the change of the cAMP concentration by a test compound maybe easily decided, when a compound (such as forskolin) capable ofincreasing the cAMP concentration coexists. It may be carried out, forexample, under the conditions described in Example 4 or 5.

Further, as a control, the transformant for cAMP detection is broughtinto contact with the platelet ADP receptor P2T_(AC) agonist in theabsence of a test compound, and then it is necessary to confirm a degreeof the decrease in the intracellular cAMP concentration therein by theagonist.

4) The GTPγS Binding-type Detecting Method

In the GTPγS binding-type detecting method of the present invention, itmay be detected whether or not a test compound is an antagonist oragonist against the platelet ADP receptor P2T_(AC) by a GTPγS bindingmethod (Lazareno, S. and Birdsall, N. J. M., Br. J. Pharmacol., 109,1120-1127, 1993), by using the polypeptide for a detecting tool, a cellmembrane fraction comprising the polypeptide, or a transformantexpressing the polypeptide, for example, in accordance with thefollowing procedure.

A cell membrane expressing the polypeptide for a detecting tool is mixedwith ³⁵S-labeled GTPγS (400 pmol/L) in a mixed solution of 20 mmol/LHEPES (pH 7.4), 100 mmol/L NaCl, 10 mmol/L MgCl₂, and 50 mmol/L GDP.After incubating in the presence of a test compound, and in the absenceof the test compound, each reaction liquid is filtered by using a glassfilter or the like, and then the radioactivity of remaining GTPγS on thefilter is measured by using a liquid scintillation counter or the like.It may be detected whether or not the test compound is an agonistagainst the ADP receptor P2T_(AC) by using the specific increase of theGTPγS binding in the presence of the test compound as an indicator.Further, it may be detected whether or not the test compound is anantagonist against the ADP receptor P2T_(AC) by using the suppression inthe increase of the GTPγS binding by a platelet ADP receptor P2T_(AC)ligand (such as 2MeSADP or ADP) in the presence of the test compound, asan indicator.

(3) The Screening Method for an Antiplatelet Agent

A ligand, antagonist, or agonist against the platelet ADP receptorP2T_(AC) may be screened by using the screening tool (including both thepolypeptide-type screening tool for an antiplatelet agent and thetransformant-type screening tool for an antiplatelet agent) of thepresent invention for an antiplatelet agent.

As previously described, ADP is known as an important factor whichinduces or promotes activation, adhesion, and aggregation of theplatelet. Further, it is considered that ADP activates the platelet viaa G protein-coupled ADP receptor P2T located in the platelet membrane(Biochem. J., 336, 513-523, 1998). Furthermore, it is considered thatTiclopidine or Clopidogrel, which is a known antiplatelet agent,functions by inhibiting the ADP receptor P2T_(AC) via its metabolite ina body (Savi, P. J., Pharmaclo. Exp. Ther., 269, 772-777, 1994). It isshown that ARL67085 or derivatives thereof (ATP analogues), derivativesof Ap4A [P¹,P⁴-di(adenosine-5′)tetraphosphate], or the like exhibit anactivity suppressing the platelet aggregation by ADP, by the P2T_(AC)antagonist activity (Mills, D. C., Thromb. Hemost., 76, 835-856, 1996;Humphries, R. G., Trends Pharmacol. Sci., 16, 179-181, 1995; and Kim, B.K., Proc. Natl. Acad. Sci. USA, 89, 2370-2373, 1992). As shown inExample 7, ADP receptor P2T_(AC) antagonists (such as 2MeSAMP orAR-C69931MX) exhibit the antiplatelet activity.

From the above information, the ligand or antagonist against theplatelet ADP receptor P2T_(AC) is useful as a substance capable ofcontrolling the activation, adhesion, and aggregation of the platelet.Therefore, the above-mentioned polypeptide for a screening tool per se,or the transformant for a screening tool per se may be used forscreening an antiplatelet agent. Namely, the polypeptide for a screeningtool per se, or the transformant for a screening tool per se may be usedfor a screening tool.

Compounds to be tested which may be screened by using the screening toolof the present invention for an antiplatelet agent are not particularlylimited, but there may be mentioned, for example, various knowncompounds (including peptides) registered in chemical files, compoundsobtained by combinatorial chemistry techniques (Terrett, N. K. et al.,Tetrahedron, 51, 8135-8137, 1995), or random peptides prepared byemploying a phage display method (Felici, F. et al., J. Mol. Biol., 222,301-310, 1991) or the like. In addition, culture supernatants ofmicroorganisms, natural components derived from plants or marineorganisms, or animal tissue extracts may be used as the test compoundsfor screening. Further, compounds (including peptides) obtained bychemically or biologically modifying compounds (including peptides)selected by the screening tool of the present invention for anantiplatelet agent may be used.

The screening methods of the present invention may be mainly dividedinto the following four groups in accordance with the detecting methodsto be used. By using any one of the detecting methods, or a combinationthereof, a substance which is useful as an antiplatelet agent can bescreened by detecting whether or not a test compound is a ligand,antagonist, or agonist against the ADP receptor P2T_(AC), and thenselecting an antagonist among test compounds. The screening methods ofthe present invention, i.e.,

-   1) a method for screening a ligand against the platelet ADP receptor    P2T_(AC) (hereinafter referred to as ligand screening method);-   2) a method for screening an antagonist or agonist against the    platelet ADP receptor P2T_(AC) by the use of changes of an    intracellular Ca²⁺ concentration in the transformant as an indicator    (hereinafter referred to as Ca²⁺-type screening method);-   3) a method for screening an antagonist or agonist against the    platelet ADP receptor P2T_(AC) by the use of changes of an    intracellular cAMP concentration in the transformant as an indicator    (hereinafter referred to as cAMP-type screening method); and-   4) a method for screening an antagonist or agonist against the    platelet ADP receptor P2T_(AC) by the use of a GTPγS binding method    (hereinafter referred to as GTPγS binding-type screening method)    will be explained in this order hereinafter.    1) The Ligand Screening Method

The ligand screening method of the present invention is not particularlylimited, so long as it comprises the steps of:

-   detecting whether or not a test compound is an ADP receptor P2T_(AC)    ligand by using the ligand detecting method of the present    invention, and-   selecting the ADP receptor P2T_(AC) ligand.

An ADP receptor P2T_(AC) ligand can be screened by the use of thebinding inhibition of the radioactive ligand obtained by the liganddetecting method as an indicator. For example, a test compound isreacted under the conditions described in Example 6 for a predeterminedtime, and then a test compound in which IC₅₀ is 10 μM or less(preferably 1 μM or less) may be selected as a ligand by the use of thebinding inhibition of [³H]-2MeSADP as an indicator.

A substance useful as an antiplatelet agent may be screened by applyingthe ligand which was screened by the ligand screening method of thepresent invention to the following Ca²⁺-type screening method, cAMP-typescreening method, and/or GTPγS binding-type screening method, and thenselecting an antagonist.

2) The Ca²⁺-type Screening Method

The Ca²⁺-type screening method of the present invention is notparticularly limited, so long as it comprises the steps of:

-   detecting whether or not a test compound is an ADP receptor P2T_(AC)    antagonist or agonist by using the Ca²⁺-type detecting method of the    present invention, and-   selecting the ADP receptor P2T_(AC) antagonist or agonist.

An agonist can be screened by the use of the increase of anintracellular Ca²⁺ concentration by a test compound in the transformantfor Ca²⁺-type detection as an indicator in the Ca²⁺-type detectingmethod.

An antagonist can be screened by an indicator in which the increase ofan intracellular Ca²⁺ concentration in the transformant for Ca²⁺-typedetection by a platelet ADP receptor P2T_(AC) agonist (such as 2MeSADPor ADP) is inhibited or suppressed by a test compound in the Ca²⁺-typedetecting method.

For example, a test compound is reacted under the conditions describedin Example 3 for a predetermined time, and then a test compound in whichIC₅₀ is 10 μM or less (preferably 1 μM or less) may be selected as asubstance exhibiting the antagonist activity by the use of inhibition ofthe increase in an intracellular Ca²⁺ concentration by 2MeSADP or ADP asan indicator.

A substance useful as an antiplatelet agent may be screened by screeningan antagonist by using the Ca²⁺-type screening method of the presentinvention.

3) The cAMP-type Screening Method

The cAMP-type screening method of the present invention is notparticularly limited, so long as it comprises the steps of:

-   detecting whether or not a test compound is an ADP receptor P2T_(AC)    antagonist or agonist by using the cAMP-type detecting method of the    present invention, and-   selecting the ADP receptor P2T_(AC) antagonist or agonist.

An agonist can be screened by the use of the decrease of anintracellular cAMP concentration by a test compound in the transformantfor cAMP-type detection as an indicator in the cAMP-type detectingmethod.

An antagonist can be screened by an indicator in which the decrease ofan intracellular cAMP concentration in the transformant for cAMP-typedetection by a platelet ADP receptor P2T_(AC) agonist (such as 2MeSADPor ADP) is inhibited or suppressed by a test compound in the cAMP-typedetecting method.

For example, a test compound is reacted under the conditions describedin Example 4 or 5 for a predetermined time, and then a test compound inwhich IC₅₀ is 10 μM or less (preferably 1 μM or less) may be selected asa substance exhibiting the antagonist activity by the use of inhibitionof the decrease in an intracellular cAMP concentration by 2MeSADP or ADPas an indicator.

A substance useful as an antiplatelet agent may be screened by screeningan antagonist by using the cAMP-type screening method of the presentinvention.

4) The GTPγS Binding-type Screening Method

The GTPγS binding-type screening method of the present invention is notparticularly limited, so long as it comprises the steps of:

-   detecting whether or not a test compound is an ADP receptor P2T_(Ac)    antagonist or agonist by using the GTPγS binding-type detecting    method of the present invention, and-   selecting the ADP receptor P2T_(AC) antagonist or agonist.

An agonist can be screened by the use of the increase of the specificGTPγS binding by a test compound as an indicator in the GTPγSbinding-type detecting method.

An antagonist can be screened by an indicator in which the increase ofthe GTPγS binding by a platelet ADP receptor P2T_(AC) agonist (such as2MeSADP or ADP) is suppressed by a test compound.

A substance useful as an antiplatelet agent may be screened by screeningan antagonist by using the GTPγS binding-type screening method of thepresent invention.

(4) Manufacture of the Pharmaceutical Composition for Antiplatelet

The present invention includes an antiplatelet agent containing, as anactive ingredient, an ADP receptor P2T_(AC) antagonist (for example,compounds, peptides, antibodies, or antibody fragments) selected by thescreening methods 2) to 4) or by a combination of the screeningmethods 1) or 4).

It may be confirmed whether the selected ADP receptor P2T_(AC)antagonist exhibits the antiplatelet activity by detecting theinhibiting activity of human platelet aggregation, for example, by themethod described in Example 7.

Further, the present invention includes a method for manufacturing apharmaceutical composition for antiplatelet comprising the steps of:

-   detecting, in a quality control test of a pharmaceutical composition    for antiplatelet, whether or not it is an ADP receptor P2T_(AC)    ligand, antagonist, or agonist by the ligand detecting method,    Ca²⁺-type detecting method, cAMP-type detecting method, and/or GTPγS    binding-type detecting method, and-   preparing a medicament.

The preparation of the present invention containing the ADP receptorP2T_(AC) antagonist (for example, compounds, peptides, antibodies, orantibody fragments) as an active ingredient may be prepared usingcarriers, fillers, and/or other additives generally used in thepreparation of medicaments, in accordance with the active ingredient.

Examples of administration include oral administration by tablets,pills, capsules, granules, fine granules, powders, oral solutions andthe like, and parenteral administration by injections (e.g.,intravenous, intramuscular, or the like), suppositories, transdermalpreparations, transmucosal absorption preparations and the like.Particularly, in the case of peptides which are digested in the stomach,a parenteral administration such as intravenous injection or the like,or preparation techniques in which the polypeptide is not digested, suchas a preparation technique disclosed in the WO95/28963 pamphlet, ispreferable.

In the solid composition for use in the oral administration, one or moreactive substances may be mixed with at least one inert diluent such aslactose, mannitol, glucose, microcrystalline cellulose,hydroxypropylcellulose, starch, polyvinyl pyrrolidone, or aluminummagnesium silicate. In the usual way, the composition may containadditives other than the inert diluent, such as a lubricant, adisintegrating agent, a stabilizing agent, or a solubilizing orsolubilization assisting agent. If necessary, tablets or pills may becoated with a sugar coating or a film of a gastric or enteric substance.

The liquid composition for oral administration may include, for example,emulsions, solutions, suspensions, syrups, and elixirs, and may containa generally used inert diluent such as purified water or ethyl alcohol.The composition may contain additives other than the inert diluent, suchas moistening agents, suspending agents, sweeteners, flavors, orantiseptics.

The injections for parenteral administration may include aseptic aqueousor non-aqueous solutions, suspensions, and emulsions. Examples of thediluent for use in the aqueous solutions and suspensions includedistilled water for injection use and physiological saline. Examples ofthe diluent for use in the non-aqueous solutions and suspensions includepropylene glycol, polyethylene glycol, plant oil (e.g., olive oil),alcohols (e.g., ethanol), polysorbate 80 and the like. Such acomposition may further contain a moistening agent, an emulsifyingagent, a dispersing agent, a stabilizing agent, a solubilizing orsolubilization assisting agent, an antiseptic or the like. Thesecompositions may be sterilized, for example, by filtration through abacteria retaining filter, blending of a germicide, or irradiation.Alternatively, they may be used by first making them into sterile solidcompositions and dissolving them in sterile water or other sterilesolvents for injection use, prior to their use.

The dose is optionally decided by taking into consideration the strengthof each active ingredient selected by the aforementioned screeningmethod, or symptoms, age, sex, or the like of each patient to beadministered.

For example, in the case of oral administration, the usual dosage for anadult (60 kg in weight) is about 0.1 to 100 mg, preferably 0.1 to 50 mgper day. In the case of parenteral administration, the usual dosage isabout 0.01 to 50 mg, preferably 0.01 to 10 mg per day in the form of aninjection.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples. The procedures may beperformed in accordance with the known methods (Maniatis, T., et al.,“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY, 1982, or the like), unless otherwise specified.

Example 1 Isolation of ADP Receptor P2T_(AC) Gene HORK3

In the present example, a full-length cDNA of an ADP receptor P2T_(AC)gene HORK3 (hereinafter briefly referred to as HORK3 gene) was obtainedby the PCR method using a human brain cDNA (manufactured by Clontech) asa template in accordance with the following procedure.

More particularly, an oligonucleotide consisting of the nucleotidesequence of SEQ ID NO: 3 was used as a forward primer, and anoligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 4was used as a reverse primer. At each of the 5′-termini of the forwardand reverse primers, a nucleotide sequence containing the XbaIrecognition sequence is added. A PCR was carried out using Taq DNAPolymerase (Ex Taq DNA polymerase; manufactured by Takara-shuzo) in thepresence of 5% dimethyl sulfoxide (DMSO). In the PCR, a cycle consistingof treatments at 94° C. for 20 seconds, 58° C. for 20 seconds, and 74°C. for 1.5 minutes was repeated 5 times, a cycle consisting oftreatments at 94° C. for 20 seconds, 55° C. for 20 seconds, and 74° C.for 1.5 minutes was repeated 5 times, and a cycle consisting oftreatments at 94° C. for 20 seconds, 50° C. for 20 seconds, and 74° C.for 1.5 minutes was repeated 25 times. As a result, a DNA fragment ofapproximately 1.0 kbp was amplified. The DNA fragment was digested withXbaI, and inserted into the XbaI site of a plasmid pEF-BOS-dhfr(Mizushima, S. and Nagata, S., Nucleic Acids Res., 18, 5322, 1990) toobtain a plasmid pEF-BOS-dhfr-HORK3.

The nucleotide sequence of the HORK3 gene in the plasmidpEF-BOS-dhfr-HORK3 was determined using a DNA sequencer (ABI377 DNASequencer; manufactured by Applied Biosystems) by a dideoxy terminatormethod. The nucleotide sequence of the HORK3 gene was that of SEQ ID NO:1.

The nucleotide sequence of SEQ ID NO: 1 contains an open reading frame(ORF) consisting of 1029 bases. The amino acid sequence (342 aminoacids) deduced from the ORF was that of SEQ ID NO: 2.

Example 2 Confirmation of Expression Distribution of HORK3 Gene inHemocytes

Blood was collected from a healthy volunteer by using heparin, and wascentrifuged at 400×g for 10 minutes. The upper layer was taken asplatelet-rich plasma.

To the lower layer, ⅓ volume of 6% dextran/physiological saline wasadded, and then the whole was allowed to stand at room temperature for 1hour. The supernatant was taken and centrifuged at 150×g for 5 minutes,and then the pellet was suspended in HBSS (Hanks' Balanced SoltSolution). The suspension was layered on an equal volume of Ficoll(Ficoll Paque; Pharmacia), and then the whole was centrifuged at 400×gfor 30 minutes. The resulting intermediate layer and pellet were takenas “a mononuclear cell fraction” and polymorphonuclear leukocytes,respectively.

CD16 microbeads (manufactured by Daiichi Pure Chemicals) were added tothe polymorphonuclear leukocytes, and then “a neutrophil fraction” wasseparated from “an eosinophil fraction” by using a magnetic stand.

EDTA was added to the previously obtained platelet-rich plasma to afinal concentration of 2 mmol/L, and then the whole was centrifuged at2500×g for 15 minutes. The resulting pellet was resuspended in 120mmol/L NaCl/2 mmol/L EDTA/30 mmol/L Tris-HCl (pH7.4), and then thesuspension was centrifuged at 2500×g for 15 minutes to obtain the pelletas “a platelet fraction”. The mononuclear cell fraction, neutrophilfraction, and eosinophil fraction were respectively washed withphysiological saline.

Total RNAs were purified from the mononuclear cell fraction, neutrophilfraction, eosinophil fraction, and platelet fraction by using acommercially available total RNA purifying reagent (isogen; manufacturedby Nippon Gene), respectively. The total RNA (5 μg) derived from eachfraction was reacted with DNase (manufactured by Nippon Gene) at 37° C.for 15 minutes. The DNase-treated total RNA was converted to cDNA by aSuperscript first-strand system (for RT-PCR; manufactured by GIBCO).

An analysis of an expression level of HORK3 mRNA in hemocytes wascarried out by using each of the above cDNAs as a template and asequence detector (Prism7700 Sequence Detector; manufactured by AppliedBiosystems). An oligonucleotide consisting of the base sequence of SEQID NO: 5 and an oligonucleotide consisting of the base sequence of SEQID NO: 6 were used as a primer set. An oligonucleotide consisting of thenucleotide sequence of SEQ ID NO: 7, in which the 5′-terminus thereofwas labeled with a fluorescence indicator FAM (6-carboxy-fluorescein)and the 3′-terminus thereof was labeled with a fluorescence indicatorTAMRA (6-carboxy-tetramethyl-rhodamine), was used as a probe whichspecifically recognizes the HORK3 cDNA.

The PCR was carried out by using an commercially available PCR reagent(TaqMan PCR core reagent; manufactured by Applied Biosystems) andrepeating a cycle consisting of treatments at 95° C. for 15 seconds and60° C. for 60 seconds 50 times in the presence of 5% DMSO. Further, toobtain a standard curve for calculating an expression level of mRNA, aPCR was carried out under the same conditions by using a human genomicDNA as a template and the above primer set and probe [J. Neurosci.Methods. 98, 9-20 (2000)].

Furthermore, to calculate, as an internal standard, an expression levelof human β-actin, a PCR was carried out under the same conditions, byusing the above each cDNA or human genomic DNA as a template, anoligonucleotide consisting of the base sequence of SEQ ID NO: 8 and anoligonucleotide consisting of the base sequence of SEQ ID NO: 9 as aprimer set, and an oligonucleotide consisting of the nucleotide sequenceof SEQ ID NO: 10, in which the 5′-terminus thereof was labeled with afluorescence indicator FAM and the 3′-terminus thereof was labeled witha fluorescence indicator TAMRA, as a probe specifically recognizingβ-actin.

Expression levels of β-actin mRNA in the mononuclear cell fraction,neutrophil fraction, eosinophil fraction, and platelet fraction were15000 copies, 19000 copies, 25000 copies, and 33000 copies per 1 ng oftotal RNA, respectively. In contrast, it was found that HORK3 mRNA wasspecifically expressed in platelets, but little expressed in mononuclearcells, neutrophils, and eosinophils.

Example 3 Confirmation of Increase in Intracellular Ca²⁺ Concentrationby 2MeSADP or ADP in C6-15 Cell Co-expressing HORK3 Protein and Gqi

When most purines such as ADP, 2MeSADP (2-methylthio-ADP), or the likeare added to a cell, the increase in intracellular Ca²⁺ via anendogenous cellular membrane receptor is observed. Therefore, to analyzewhether or not a protein derived from a gene introduced exogenouslyreacts with ADP or 2MeSADP, it is preferable to express the protein byusing a cell which does not react with ADP or 2MeSADP. It is known thatC6-15, a rat glioma cell line, does not react with ADP or the like(Change, K. et al., J. Biol. Chem., 270, 26152-26158, 1995). In thepresent example, the C6-15 cell was used to express the HORK3 protein.

Further, the plasmid pEF-BOS-dhfr-HORK3 obtained in Example 1 was usedas an expression plasmid to be used for expressing the HORK3 protein.

An expression vector for expressing the chimeric protein of Gq and Giused in the present example was prepared in accordance with a method ofConklin, B. R. et al. (Nature, 363, 274-276, 1993) by cloning a gene(hereinafter referred to as Gqi gene), which had been constructed bysubstituting five amino acids (Glu-Tyr-Asn-Leu-Val; the amino acidsequence of SEQ ID NO: 12) at the C-terminus of Gq with five amino acids(Asp-Cys-Gly-Leu-Phe; the amino acid sequence of SEQ ID NO: 11) at theC-terminus of Gi, into the plasmid pEF-BOS. The constructed plasmid wasnamed plasmid pEF-BOS-Gqi.

The C6-15 cells were seeded on a 96-well plate (96 well Black/clearbottom plate, collagen I coated; manufactured by BECTON DICKINSON) sothat the concentration of cells became 2×10⁴ cells/well. After culturingfor 48 hours, a gene transfection was carried out by using atransfection reagent (LipofectAMINE 2000; manufactured by GIBCO BRL) andthe combination of the plasmid pEF-BOS-dhfr-HORK3 (50 ng/well) and theplasmid pEF-BOS-Gqi (50 ng/well). As a control, a gene transfection wascarried out by using the combination of the plasmid pEF-BOS-dhfr (i.e.,an empty vector without the HORK3 gene) and the plasmid pEF-BOS-Gqi,instead of the combination of the plasmid pEF-BOS-dhfr-HORK3 and theplasmid pEF-BOS-Gqi.

After 24 hours from the gene transfection procedure, the medium wasdiscarded, and then HBBS (Hanks' Balanced Solt Solution; 100 μL/well)containing 4 μmol/L Fluo-3, AM (manufactured by Molecular Probe), 0.004%pluronic acid (trademark=Pluronic F127, manufactured by MolecularProbe), 1% fetal bovine serum (FBS), 20 mmol/L HEPES, and 2.5 mmol/Lprobenecid was added. After incubating at 37° C. for 1 hour, cells werewashed with HBBS (manufactured by GIBCO) containing 20 mmol/L HEPES and2.5 mmol/L probenecid 4 times, and then HBBS (100 μL/well) containing 20mmol/L HEPES and 2.5 mmol/L probenecid was added.

A time course of the change in the intracellular Ca²⁺ concentration wasmeasured by using FLIPR (manufactured by Molecular Device). Moreparticularly, after 10 seconds from the beginning of measurement,2MeSADP or ADP was added to wells to a final concentration of 3×10⁻⁵mol/L to 1×10⁻¹² mol/L. After the addition, each fluorescence intensitywas measured at an interval of 1 second for 50 seconds, and then at aninterval of 6 seconds for 4 minutes.

The increase of the intracellular Ca²⁺ concentration dependent upon theconcentration of 2MeSADP or ADP was observed in the cells to which thecombination of the plasmid pEF-BOS-dhfr-HORK3 and the plasmidpEF-BOS-Gqi had been transfected. In contrast, the change of theintracellular Ca²⁺ concentration by 2MeSADP or ADP was not observed inthe cells to which the combination of the plasmid pEF-BOS-dhfr (emptyvector) and the plasmid pEF-BOS-Gqi had been transfected.

The change of the intracellular Ca²⁺ concentration by 2MeSADP or ADP inthe cells to which the plasmid pEF-BOS-dhfr-HORK3 and the plasmidpEF-BOS-Gqi had been transfected was measured, each peak value invarious concentrations of 2MeSADP or ADP was plotted, and then thedependency upon the concentration was analyzed by using a Logisticregression method. As a result, it was found that EC₅₀ of 2MeSADP was5.4 nmol/L, and that EC₅₀ of ADP was 220 nmol/L. As described above, itwas confirmed that the dose-dependent change of the intracellular Ca²⁺concentration was induced by reacting with 2MeSADP or ADP in thetransformant for Ca²⁺-type detection co-expressing the polypeptide for adetecting tool and Gqi.

As described above, it was found that the HORK3 protein, one of thepolypeptides for detection, is a Gi-coupled receptor which reacts withADP. Further, as apparent from the results in the present example, ithas become feasible to screen an agonist or antagonist by measuring thechange of the intracellular Ca²⁺ concentration in the C6-15 cellco-expressing the HORK3 protein (one of the polypeptides for a detectingtool) and Gqi.

Example 4 Confirmation of Inhibition of cAMP Production by 2MeSADP orADP in CHO Cell Expressing HORK3 Protein

It was found from Example 3 that the ADP receptor was a Gi-coupledreceptor, and thus it is expected that the ADP receptor has an activityof suppressing the adenylate cyclase activity. Therefore, when thecAMP-type screening method of the present invention is carried out, itis preferable to select a cell line not having an activity ofsuppressing the adenylate cyclase activity by ADP or 2MeSADP, as a hostcell to be used for expressing the ADP receptor protein. It was foundthat the CHO cell was the most preferable by searching for a cell inwhich an amount of cAMP produced by a forskolin stimulus was notdecreased by ADP or 2MeSADP in accordance with the following procedure,and thus the CHO cell was used as the cell to be used for expressing theADP receptor protein. In this connection, a dihydrofolate reductase(DHFR; an essential enzyme for de novo synthesis of nucleic acids)defective cell line [CHO-dhfr(−) line] was used in the present example.The plasmid pEF-BOS-dhfr-HORK3 was used as an expression plasmid to beused for expressing the HORK3 protein.

The CHO-dhfr(−) line was seeded on each 10 cm-petri dish (1×10⁶cells/dish) by using an αMEM (with nucleic acids) medium. Afterculturing for 1 day, a gene transfection was carried out by using atransfection reagent (LipofectAMINE 2000; manufactured by GIBCO BRL) andthe plasmid pEF-BOS-dhfr-HORK3 (8 μg). As a control, a gene transfectionwas carried out by using the plasmid pEF-BOS-dhfr (i.e., an empty vectorwithout the HORK3 gene), instead of the plasmid pEF-BOS-dhfr-HORK3.

After 24 hours from the gene transfection procedure, the transfectedcells were taken and suspended in an αMEM (without nucleic acids) mediumcontaining 100 nmol/L methotrexate (a competitive inhibitor of DHFR;manufactured by Wako Pure Chemical Industries). Each suspension wasgradually diluted and reseeded on each 10 cm-petri dish. Colonies whichappeared after 2 weeks were individually taken, and used as a CHO cellexpressing the HORK3 protein or a control CHO cell transfected with theempty vector in the following experiment.

The CHO cells expressing the HORK3 protein or the CHO cells transfectedwith the empty vector were seeded on a 24-well plate (1×10⁵ cells/well).After culturing for 1 day, cells were treated with an αMEM (withoutnucleic acids) medium containing 1 mmol/L 3-isobutyl-1-methylxanthine(manufactured by Sigma) and 0.1% BSA for 10 minutes, and then thecombination of 3 μmol/L forskolin (manufactured by Wako Pure ChemicalIndustries) and 2MeSADP (a final concentration=1×10⁻¹² to 1×10⁻⁷ mol/L),or the combination of 3 μmol/L forskolin and ADP (a finalconcentration=1×10⁻¹⁰ to 1×10⁻⁵ mol/L) was added dropwise. Afterincubating at 37° C. for 30 minutes, the culture supernatant wasdiscarded, and then cells were dissolved in a cell lysis reagentcontained in a cAMP-EIA system (BIOTRAK; manufactured by Amersham).

The amount of cAMP produced in each cell under each condition wasmeasured by using the cAMP-EIA system in accordance with a protocolattached thereto. When the amount of cAMP produced by stimulating with 3μmol/L forskolin alone was regarded as 100%, a concentration-dependentcurve for the amount of cAMP in the presence of 2MeSADP or ADP wasdrawn. From the concentration-dependent curve, the responsiveness of2MeSADP and ADP against the HORK3 protein were EC₅₀=0.07 nmol/L andEC₅₀=35 nmol/L, respectively.

In contrast, no changes in the amount of cAMP produced by the forskolinstimulus were observed in the CHO cell transfected with the emptyvector, even if 2MeSADP or ADP was added.

Example 5 Effects of Inhibitors and Confirmation of Inhibition of cAMPProduction by 2MeSADP or ADP in C6-15 Cell Expressing HORK3 Protein

It was found that the C6-15 cell was also the most preferable as a cellin which the amount of cAMP produced by the forskolin stimulus was notdecreased by ADP or 2MeSADP, and thus the C6-15 cell was also used asthe cell to be used for expressing the ADP receptor protein.

The plasmid pEF-BOS-dhfr-HORK3 was used as an expression vector to beused for expressing the HORK3 protein.

The C6-15 cells were seeded on each 10 cm-petri dish (1×10⁶ cells/dish)by using a DMEM medium. After culturing for 1 day, an gene transfectionwas carried out by using a transfection reagent (LipofectAMINE 2000;manufactured by GIBCO BRL), and the plasmids pEF-BOS-dhfr-HORK3 (8 μg)and pEF-BOS-neo (Nucleic Acid Res., 18, 5322, 1990; 0.8 μg).

After 24 hours from the gene transfection procedure, the transfectedcells were taken and suspended in a DMEM medium containing 0.6 mg/mLG418 (manufactured by GIBCO BRL). Each suspension was gradually dilutedand reseeded on each 10 cm-petri dish. Colonies which appeared after 2weeks were individually taken, and used as a C6-15 cell expressing theHORK3 protein in the following experiment.

The C6-15 cells expressing the HORK3 protein were seeded on a 96-wellplate (1×10⁴ cells/well). After culturing for 1 day, cells were treatedwith a DMEM medium containing 1 mmol/L 3-isobutyl-1-methylxanthine(manufactured by Sigma) and 0.1% BSA for 10 minutes, and then thecombination of 1 μmol/L forskolin (manufactured by Wako Pure ChemicalIndustries) and 2MeSADP (a final concentration=1×10⁻¹² to 1×10⁻⁷ mol/L),or the combination of 1 μmol/L forskolin and ADP (a finalconcentration=1×10⁻¹⁰ to 1×10⁻⁵ mol/L) was added dropwise. Afterincubating at 37° C. for 30 minutes, the culture supernatant wasdiscarded, and then cells were dissolved in PBS containing 0.2% TritonX-100.

The amount of cAMP produced in each cell under each condition wasmeasured by using a cAMP HTRF kit (manufactured by CIS biointernational) in accordance with a protocol attached thereto. When theamount of cAMP produced by stimulating with 1 μmol/L forskolin alone wasregarded as 100%, a concentration-dependent curve for the amount of cAMPin the presence of 2MeSADP or ADP was drawn. From theconcentration-dependent curve, the responsiveness of 2MeSADP and ADPagainst the HORK3 protein were EC₅₀=0.08 nmol/L and EC₅₀=42 nmol/L,respectively. The C6-15 cell expressing the HORK3 protein exhibitedalmost the same property as that of the CHO cell expressing the HORK3protein described in Example 4.

Next, effects of compounds, which are known to suppress the plateletaggregation, on the suppression of the amount of cAMP produced by theforskolin stimulus in the C6-15 cell expressing the HORK3 protein by2MeSADP were examined. As the compounds suppressing the plateletaggregation, 2MeSAMP (2-methylthio-adenosine monophosphate) (Thromb.Haemost., 81, 111-117, 1999) or AR-C69931MX(N6-[2-methylthioethyl]-2-[3,3,3-trifluoropropylthio]-5′-ade nylic acid,monoanhydride with dichloromethylenebiphosphonic acid) (J. Med. Chem.,42, 213-220, 1999) was used. In the above measurement system, cells weretreated with a solution [prepared by dissolving 2MeSAMP (10⁻⁷ to 10⁻⁴mol/L) or AR-C69931MX (10⁻¹² to 10⁻⁶ mol/L) in DMEM medium containing 1mmol/L 3-isobutyl-1-methylxanthine (manufactured by Sigma) and 0.1% BSA]for 10 minutes, and then the combination of 1 μmol/L forskolin(manufactured by Wako Pure Chemical Industries) and 10 nmol/L 2MeSADPwas added dropwise. After incubating at 37° C. for 30 minutes, theculture supernatant was discarded, and then cells were dissolved in PBScontaining 0.2% Triton X-100. The amount of cAMP produced was measuredby using the cAMP HTRF kit in accordance with a protocol attachedthereto. When the amount of cAMP produced by stimulating with 1 μmol/Lforskolin alone was regarded as 0%, and that by stimulating with thecombination of 1 μmol/L forskolin and 10 nmol/L 2MeSADP was regarded as100%, a concentration-dependent curve of each inhibitor was drawn. Fromthe concentration-dependent curve, inhibitory effects of 2MeSAMP andAR-C69931MX on the suppression by 2MeSADP in the amount of cAMP producedby the forskolin stimulus were IC₅₀=5 μmol/L and IC₅₀=2.4 nmol/L,respectively.

Example 6 Binding Assay of 2MeSADP to C6-15 Cell Expressing HORK3Protein

The C6-15 cells expressing the HORK3 protein prepared in Example 5 weretaken and washed, and then suspended in 20 mmol/L Tris-HCl (pH7.4)containing 5 mmol/L EDTA and a protease inhibitor cocktail set(Complete™; manufactured by Boeringer Mannheim). The whole washomogenized by using a POLYTRON, and ultracentrifuged. The pellet wassuspended in 50 mmol/L Tris-HCl (pH7.4) containing 1 mmol/L EDTA, 100mmol/L NaCl, 0.1% BSA, and Complete™ to obtain a membrane fraction.

After [³H]-2MeSADP (manufactured by Moravek Biochemical) was added to 20μg of the membrane fraction as a final concentration of 0.25 to 50nmol/L, and was incubated in 250 μL of 50 mmol/L Tris-HCl (pH7.4)containing 1 mmol/L EDTA, 100 mmol/L NaCl, 0.1% BSA, and Complete™ atroom temperature for 1 hour, the whole was collected on a glass filterby using a cell harvester. A microscintillator was added to the glassfilter, and then a total amount of binding to the membrane fraction wasmeasured by using a liquid scintillation counter. Further, an amount ofnon-specific binding to the membrane fraction was measured by adding2MeSADP (a final concentration=50 μmol/L) in the above assay. As aresult, it was found that [³H]-2MeSADP was specifically bound to themembrane fraction of the C6-15 cell expressing the HORK3 protein. As aresult of the Scatchard analysis for the binding, the dissociationconstant of the [³H]-2MeSADP binding to the membrane fraction of theC6-15 cell expressing the HORK3 protein was Kd=0.27 nmol/L, and themaximum binding was Bmax=3.7 pmol/mg. In contrast, such a specificbinding was not observed in the membrane fraction of the host cell C6-15not expressing the HORK3 protein.

Next, an effect of 2MeSAMP or AR-C69931MX was examined by using themembrane fraction of the C6-15 cell expressing the HORK3 proteinprepared in Example 6, and by using an activity of inhibiting the[³H]-2MeSADP binding as an indicator. More particularly, after 2MeSAMP(10⁻⁷ to 10⁻⁴ mol/L) or AR-C69931MX (10⁻¹² to 10⁻⁶ mol/L) and 1 nmol/L[³H]-2MeSADP were added to 20 μg of the membrane fraction from the C6-15cell expressing the HORK3 protein, and were incubated at roomtemperature for 2 hours, the whole was collected on a glass filter byusing a cell harvester. A microscintillator was added to the glassfilter, and then radioactivity was measured by using a liquidscintillation counter. Further, radioactivity was measured by not addingthe compound and by adding 2MeSADP (a final concentration=10 μmol/L) inthe above assay, as a total amount of binding and an amount ofnon-specific binding, respectively. From the concentration-dependentcurve of each compound, the inhibitory effects of 2MeSAMP andAR-C69931MX on the binding of 2MeSADP to the HORK3 protein were IC₅₀=4.9μmol/L and IC₅₀=9.8 nmol/L, respectively.

In addition to the results in Example 3, the amount of cAMP produced bythe forskolin stimulus in the CHO and C6-15 cells expressing the HORK3protein was suppressed by 2MeSADP or ADP in Examples 4 and 5,respectively. These results made it clearer that the ADP receptor wascoupled to Gi. Further, from the results in Examples 4 and 5, it hasbecome feasible to screen an agonist or antagonist by measuring thechange of the intracellular cAMP concentration in the CHO or C6-15 cellexpressing the HORK3 protein (one of the polypeptides for a screeningtool). Furthermore, it is possible to screening a ligand by measuringthe inhibition of 2MeSADP binding to the membrane fraction of the C6-15cell expressing the HORK3 protein, in accordance with the methoddescribed in Example 6.

As described above, the HORK3 protein is a Gi-coupled receptor expressedin platelets, from the results in Examples 2 to 6, and thus it isconsidered that the HORK3 protein is an entity of the platelet ADPreceptor P2T_(AC) in which the existence thereof has been suggested.

Example 7 Confirmation of Activity of Inhibiting Human PlateletAggregation

Blood was collected from a healthy person (adult, male) by using 1/10volume of sodium citrate, and then platelet-rich plasma(PRP) wasprepared in accordance with a method of De Marco et al. (J. Clin.Invest., 77, 1272-1277, 1986). The PRP was used after preparing it to3×10⁸ cells/mL by using an automatic blood cell counter (MEK6258; NihonKohden Corporation). As an inducer of aggregation, ADP which is aproduct manufactured by MC Medical Corporation was used. Further,2MeSAMP or AR-C69931MX was used, and physiological saline was used as asolvent to be used for dissolving the compounds. In this connection, itwas confirmed in Example 5 that 2MeSAMP or AR-C69931MX exhibited anactivity as a HORK3 protein antagonist.

The platelet aggregation was measured by using an aggregometer (MCM HemaTracer 212; MC Medical Corpration). More particularly, PRP (80 μL) and asample (2MeSAMP or AR-C69931MX) or the solvent (10 μL) were incubated at37° C. for 1 minute, and then 10 μL of ADP (20 to 200 μmol/L) was added.Changes of a transmitted light were recorded for 10 minutes, and then anaggregation inhibition (%) was calculated from the maximum aggregationrate.

The results when 2MeSAMP and AR-C69931MX were used are shown in FIGS. 1and 2, respectively. The data for 2MeSAMP (FIG. 1) are represented bythe “average” of the experiment results obtained by using twovolunteers. The data for AR-C69931MX (FIG. 2) are represented by the“average+standard error” of the experiment results obtained by usingthree volunteers. Both agents inhibited the ADP-induced plateletaggregation concentration-dependently. The inhibitory strength wasdependent on the concentration of ADP as an inducer.

It is apparent from the present example that an HORK3 protein antagonistexhibits an antiplatelet activity.

INDUSTRIAL APPLICABILITY

A substance inhibiting the platelet ADP receptor P2T_(AC) activityexhibits an antiplatelet activity, and makes it possible to treat athrombotic disorders.

Therefore, according to the screening tool of the present invention foran antiplatelet agent, it is possible to screen and evaluate a plateletADP receptor P2T_(AC) antagonist which is useful as an antiplateletagent.

It is possible to select a platelet ADP receptor P2T_(AC) antagonist andto screen a substance which is useful as an antiplatelet agent, by usingthe detecting method of the present method for an antagonist or agonist,or by using the combination of the ligand detecting method of thepresent invention and the detecting method of the present method for anantagonist or agonist.

Further, it is possible to manufacture a pharmaceutical composition forantiplatelet by preparing a medicament by using the substance selectedby the above screening method as an active ingredient, and carriers,fillers, and/or other additives.

Furthermore, it is possible to use the detecting method of the presentinvention for a ligand, antagonist, or agonist not only for screeningthe substance useful as an antiplatelet agent, but also as a qualitycontrol test of a pharmaceutical composition for antiplatelet.

It is possible to manufacture a pharmaceutical composition forantiplatelet, by detecting whether or not a test compound is a plateletADP receptor P2T_(AC) ligand, antagonist, or agonist using the detectingmethod of the present invention for a ligand, antagonist, or agonist,and then by preparing a medicament using the antagonist or ligand.

Free Text in Sequence Listing

Features of “Artificial Sequence” are described in the numericidentifier <223> in the Sequence Listing. More particularly, eacholigonucleotide consisting of the base sequence of SEQ ID NO: 3 or 4 isan artificially synthesized primer sequence.

As above, the present invention is explained with reference toparticular embodiments, but modifications and improvements obvious tothose skilled in the art are included in the scope of the presentinvention.

1. A method of screening for an agent that inhibits plateletaggregation, comprising the steps of: detecting whether or not acompound is an ADP receptor P2T_(AC) ligand, antagonist, or agonist byat least one method selected from (a) a method for detecting whether ornot the compound is an ADP receptor p2T_(AC) ligand, comprising thesteps of: bringing a transformant cell membrane fraction comprising atleast one of (1) a polypeptide comprising the amino acid sequence of SEQID NO: 2; (2) a polypeptide comprising an amino acid sequence in which 1to 10 amino acids are deleted, substituted, and/or added in the aminoacid sequence of SEQ ID NO: 2, and exhibiting an activity of suppressingan adenylate cyclase activity by binding to ADP and coupling with Gi; or(3) a polypeptide comprising an amino acid sequence having a 95% or morehomology with the amino acid sequence of SEQ ID NO: 2, and exhibiting anactivity of suppressing an adenylate cyclase activity by binding to ADPand coupling with Gi, or bringing a transformant which is transformedwith an expression vector comprising a DNA encoding at least one of saidpolypeptides (1) to (3),  into contact with the compound, in thepresence of a labeled ligand of an ADP receptor P2T_(AC);and analyzingthe amount of the labeled ligand which binds to the transformant cellmembrane fraction or the transformant in the presence of and in theabsence of the compound, wherein the ADP receptor P2T_(AC) ligand isidentified when the amount of labeled ligand that binds to thetransformant cell membrane fraction or the transformant is lower in thepresence of the compound than in the absence of the compound; (b) amethod for detecting whether or not the compound is an ADP receptorP2T_(AC) antagonist or agonist, comprising the steps of: bringing thecompound and optionally a known ADP receptor P2T_(AC) agonist intocontact with a transformant which is transformed with an expressionvector comprising a DNA encoding (1) a polypeptide comprising the aminoacid sequence of SEQ ID NO: 2; (2) a polypeptide comprising an aminoacid sequence in which 1 to 10 amino acids are deleted, substituted,and/or added in the amino acid sequence of SEQ ID NO: 2, and exhibitingan activity of suppressing an adenylate cyclase activity by binding toADP and coupling with Gi; or (3) a polypeptide comprising an amino acidsequence having a 95% or more homology with the amino acid sequence ofSEQ ID NO: 2, and exhibiting an activity of suppressing an adenylatecyclase activity by binding to ADP and coupling with Gi saidtransformant co-expressing a G protein chimera comprising a polypeptidehaving an activity of promoting a phospholipase C activation, said Gprotein chimera having the amino acid sequence of SEQ ID NO: 11 at theC-terminus; and analyzing the intracellular Ca²⁺ concentration in thetransformant in the presence of and in the absence of the compound,wherein if the intracellular Ca²⁺ increases in the presence of thecompound and does not increase in the presence of the compound in a cellthat does not express the polypeptide described in (1), (2) or (3), thecompound is an ADP receptor p2T_(AC) agonist and wherein if theintracellular Ca²⁺ decreases in the presence of the compound and theknown ADP receptor p2T_(AC) agonist, the compound is an ADP receptorp2T_(AC) antagonist; and (c) a method for detecting whether or not thecompound is an ADP receptor p2T_(AC) antagonist or agonist, comprisingthe steps of: bringing a transformant which is transformed with anexpression vector comprising a DNA encoding (1) a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2; (2) a polypeptide comprising anamino acid sequence in which 1 to 10 amino acids are deleted,substituted, and/or added in the amino acid sequence of SEQ ID NO: 2,and exhibiting an activity of suppressing an adenylate cyclase activityby binding to ADP and coupling with Gi; or (3) a polypeptide comprisingan amino acid sequence having a 95% or more homology with the amino acidsequence of SEQ ID NO: 2, and exhibiting an activity of suppressing anadenylate cyclase activity by binding to ADP and coupling with Gi  intocontact with the compound and optionally a known ADP receptor p2T_(AC)agonist; and analyzing the intracellular cAMP concentration in thetransformant in the presence of and in the absence of the compound,wherein if the intracellular cAMP decreases in the presence of thecompound and does not decrease in the presence of the compound in a cellthat does not express the polypeptide described in (1), (2) or (3), thecompound is an ADP receptor P2T_(AC) agonist and wherein if theintracellular cAMP does not decrease in the presence of the compound andthe known ADP receptor p2T_(AC) agonist, the compound is an ADP receptorp2T_(AC) antagonist; selecting the ADP receptor p2T_(AC) antagonist asthe agent; and confirming that the selected ADP receptor p2T_(AC)antagonist exhibits a platelet aggregation inhibition activity bydetecting inhibition of human platelet aggregation by the selected ADPreceptor p2T_(AC) antagonist.
 2. A method of screening for an agent thatinhibits platelet aggregation, comprising the steps of: detectingwhether or not a compound is an ADP receptor p2T_(AC) ligand,antagonist, or agonist by at least one method selected from (a) a methodfor detecting whether or not the compound is an ADP receptor P2T_(AC)ligand, comprising the steps of: bringing a transformant cell membranefraction comprising a polypeptide comprising the amino acid sequence ofSEQ ID NO: 2, or a transformant which is transformed with an expressionvector comprising a DNA encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO: 2, into contact with the compound, in thepresence of a labeled ligand of an ADP receptor P2T_(AC);and analyzingthe amount of the labeled ligand which binds to the transformant cellmembrane fraction or the transformant in the presence of and in theabsence of the compound, wherein the ADP receptor p2T_(AC) ligand isidentified when the amount of labeled ligand that binds to thetransformant cell membrane fraction or the transformant is lower in thepresence of the compound than in the absence of the compound; (b) amethod for detecting whether or not the compound is an ADP receptorP2T_(AC) antagonist or agonist, comprising the steps of: bringing thecompound and optionally a known ADP receptor P2T_(AC) agonist intocontact with a transformant which is transformed with an expressionvector comprising a DNA encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 2, said transformant co-expressing a G proteinchimera comprising a polypeptide having an activity of promoting aphospholipase C activation, said G protein chimera having the amino acidsequence of SEQ ID NO: 11 at the C-terminus; and analyzing theintracellular Ca²⁺ concentration in the transformant in the presence ofand in the absence of the compound, wherein if the intracellular Ca²⁺increases in the presence of the compound and does not increase in thepresence of the compound in a cell that does not express the polypeptidecomprising the amino acid sequence of SEQ ID NO: 2, the compound is anADP receptor p2T_(AC) agonist and wherein if the intracellular Ca²⁺decreases in the presence of the compound and the known ADP receptorP2T_(AC) agonist, the compound is an ADP receptor p2T_(AC) antagonist;and (c)a method for detecting whether or not the compound is an ADPreceptor P2T_(AC) antagonist or agonist, comprising the steps of:bringing a transformant which is transformed with an expression vectorcomprising a DNA encoding a polypeptide comprising the amino acidsequence of SEQ ID NO: 2, into contact with the compound and optionallya known ADP receptor P2T_(AC) agonist; and analyzing the intracellularcAMP concentration in the transformant in the presence of and in theabsence of the compound, wherein if the intracellular cAMP decreases inthe presence of the compound and does not decrease in the presence ofthe compound in a cell that does not express the polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2, the compound is an ADP receptorp2T_(AC) agonist and wherein if the intracellular cAMP does not decreasein the presence of the compound and the known ADP receptor P2T_(AC)agonist, the compound is an ADP receptor p2T_(AC) antagonist; selectingthe ADP receptor p2T_(AC) antagonist as the agent; and confirming thatthe selected ADP receptor P2T_(AC) antagonist exhibits a plateletaggregation inhibition activity by detecting inhibition of humanplatelet aggregation by the selected ADP receptor P2T_(AC) antagonist.